Abstract
Introduction: Myelofibrosis (MF) arises at the level of hematopoietic stem and progenitor cells (HSC/HPC) due to a sequence of mutational events that results in increased JAK/STAT signaling. JAK2 has served as a target for drug development and has led to the development of several novel small molecule inhibitors. Clinical trials of these agents have revealed their ability to decrease the degree of splenomegaly and to improve systemic symptoms of PMF patients, but not to substantially affect the progeny of the malignant clone or the rate of disease progression. MF HSC/HPCs and their progeny are known to elaborate a number pro-inflammatory mediators including lipocalin (LCN2) (Lu et al, Blood, 2015), and IL-8 which are capable of creating a tumor promoting microenvironment. LCN2 promotes osteoblast formation and marrow fibrosis (Lu et al, Blood, 2013) while IL8 promotes endothelial cell proliferation. We have previously reported that plasma LCN2 levels are increased in MF patients (Lu et al, ASH, 2016) and show now that plasma IL-8 levels are also dramatically increased (Figure 1). Furthermore the levels of IL-8 as shown by IHC were increased in splenic myeloid cells especially megakaryocytes and the IL-8 receptor CXCR2 was expressed at greater levels by MF splenic littoral cells that line the sinusoids as well as endothelial cells as compared to normal spleens. We hypothesize that effective treatment of MF would require a combination of drugs that directly deplete the malignant HSC/HPCs as well as dampen the tumor promoting microenvironment. In a previous study, we have found the MDM2 protein, a negative regulator of p53, are highly expressed in PV CD34+ cells from PV patients and can be targeted by nutlin therapy (Lu et al, Blood, 2013). In order to assess if nutlin therapy would deplete MF CD34+ cells, using flow cytometry method, we documented that MDM2 protein expression was significantly increased in MF CD34+ cells as compared to normal CD34+ cells and was higher than that in PV CD34+ cells (Figure 2). This finding suggests that MF malignant HSC/HPC might also be sensitive to the treatment with a nutlin. Furthermore, MF HSC/HPCs reside in the BM and extra medullary sites containing dysfunctional microenvironments which promote the predominance of the malignant stem cells. We assume that BET inhibitors might dampen the MF inflammatory milieu since BET proteins play a central role in regulation of inflammatory responses. BET proteins interact with transcription factors (TF) including NF-KB, P53 and c-Myc, and downregulate the transcriptional activation of numerous cytokines including IL-6 and IL-8. We then hypothesize that treatment with low doses of nutlin combined with JQ1, a BET inhibitor, will target MF malignant cells as well as correct MF HSC tissue specific supportive microenvironments by decreasing pro-inflammatory cytokine generation.
Results: 1. Treatment with a combination of low dose RG7112 and JQ1 selectively decreased hematopoietic colony formation by MF CD34+ cells. Exposure to either low dose RG7112 (200nM) or JQ1 (50nM) alone for 2 days increased the degree of MF CD34+ cell apoptosis modestly, while a combination of the 2 drugs significantly induced MF CD34+ cell apoptosis (p<0.05). By contrast, treatment with each drug alone or in combination however did not significant induce apoptosis of normal CD34+ cells. Combination treatment of RG7112 and JQ1 inhibited total number of colonies generated by MF CD34+ cells by 60%, as compared to the treatment with each drug alone (p=0.004 and p=0.03, respectively). Combination treatment with RG7112 and JQ1 did not, however, decrease colony formation by normal HPC. In a small number of cases, combination therapy decreased the numbers of homozygous JAK2V617F+ colonies assayed from MF CD34+ cells (at range from 8% to 20%).
2. Both RG7112 and JQ1 treatment decrease the elaboration of MF pro-inflammatory cytokines. Treatment of MF mononuclear cells (MNC) with RG7112 but not JQ1 decreased LCN2 levels in conditioned medium (CM) (at range from 5% to 39%, p=0.012); while treatment with JQ1 at 50nM strongly decreased the levels of IL-8 in MF MNC CM by 50% (p=0.0003).
Summary: These data indicate that JQ1 combined with RG7112 might be a potential effective strategy for the treatment of MF. This drug combination will not only selectively target MF HSC/HPCs but also alter the MF HSC promoting microenvironment by inhibiting the elaboration of pro-inflammatory mediators.
No relevant conflicts of interest to declare.
This feature is available to Subscribers Only
Sign In or Create an Account Close Modal